The invention concerns papillomavirus DNAs and more particularly the probes derived from these papillomaviruses, as well as the processes which use them for the in vitro diagnosis of papillomavirus infections.
The expression xe2x80x9cpapillomavirusxe2x80x9d covers a great number of viruses having in common being considered responsible for several forms of viral infection ranging from relatively benign warts of the skin or mucous membranes to hyperplasias susceptible to degenerating into intra-epithelial neoplasms or into various forms of skin cancer. To be noted also among the papillomavirus infections are particularly the epidermodysplasias verruciformis which will sometimes be referred to hereafter by the expression xe2x80x9cEVxe2x80x9d.
A certain number of types of papillomavirus have already been described. In the context of the present patent application, several new types and sub-types of papillomavirus will be described which have been isolated from warts or disseminated macular lesions, likely to lead to the development of precocious skin cancers in a large proportion of affected patients.
Recent studies have revealed the importance of immune factors and the manor role of human papillomaviruses (HPV), to these factors added are the role previously described in the literature of various genetic factors and actinic radiations in the pathogenesis of papillomavirus infections.
The invention is the result of observations regarding the relative behavior of a great number of newly isolated papillomaviruses whose essential genomic characteristics will be defined below.
The study of a small number of EV cases has already lead to the characterization of 6 types of HPV after molecular cloning of their genomes (KREMSDORF, D. et al. 1982, J. Virol. 43, 436-447, and KREMSDORF et al. 1983, J. Virol. 48, 340-351). These HPV have been divided into three groups as a function of the lack of cross-hybridization or very weak cross-hybridization between the genomes of the different groups. The first group includes the HPV3a and 10 which are associated with the plane warts observed in certain EV patients and in the general population ; DNA sequences related to those of HPV3a have been found in the cancer of an EV patient. The second group includes HPV5, 8 and 12, the genomes of HPV5 and 8 having been detected in the cancers of EV patients. With the exception of a kidney transplant recipient presenting an immuno-suppression, who turned out to be infected by HPV5, the virus of the two latter groups have been detected only among EV patients, most of them being infected by several viruses. It should be noted that among the 14 types of HPV currently mentioned in the literature (bibliographic references 1-5, 8, 9, 13, 14, 16, 18-20 indicated further on), four turn out to be specifically associated with EV which is a rare disease.
The labors which have led to this invention and which have permitted the isolation of a large number of new types and sub-types of papillomavirus, create the possibility of more highly refined in vitro diagnostic techniques. More particularly, the invention provides perfected techniques for papillomavirus identification, for example, of those obtained from lesions ox biopsy sections, and allows for more precise diagnoses which may also result in better prognoses with regard to the possible evolution of the lesions in question.
As a general rule, it should be noted that, despite being very different from each other, the papillomaviruses have sizes of the order of 7000-8000 base pairs. In addition, their genomes may nevertheless present certain degrees of homology. In what follows, reference will be made to evaluations of the percentage of homology between the various types and sub-types of papillomavirus, these homology percentages result from hybridization assays performed under conditions referred to as xe2x80x9cnon-stringentxe2x80x9d or xe2x80x9cnon-strictxe2x80x9d, or under hybridization conditions called xe2x80x9cstringentxe2x80x9d ou xe2x80x9cstrictxe2x80x9d.
Among the papillomaviruses may be distinguished several types of papillomavirus, these may be distinguished by their percentages of homology as measured under strict or stringent conditions. Papillomaviruses which, under these conditions, present homology percentages of less than 50%, belong to different types. It may be noted in this regard, that the homology percentages between viruses of different types may even fall to zero under said strict or stringent conditions. Under these same conditions, viruses showing homology percentages of more than 50% are considered as belonging to the same type and form the different syb-types within this same type.
Hybridization assays under non-strict or non-stringent conditions implies the mutual placing into contact of DNAs derived from two viral isolates under the following conditions as described by HEILMAN, C. A. et al. 1980, J. Virol. 36, 395-407, and CROISSANT et al. 1982, C. R. Acad. Sc. Paris, 294, 81-586 (hetero-duplex molecules).
Hybridization assays under strict or stringent conditions imply the placing into mutual contact of DNAs derived from two viral isolates under the conditions described by KREMSDORF, D. Et al. (1982, J. Virol. 43, 436-447, and J. Virol. 48, 340-351) and DAVIS, R. W. et al. 1971, Methods Enzymol. 21, 413-428 (hetero-duplex molecules).
Schematically, it may be noted that those papillomaviruses belonging to one same type presenting hybridizable sequences having virtually identical nucleotide sequences over 80 to 100% of the totality of their respective lengths, these homologous sequences may be reduced to 60% or less among papillomaviruses of different types. The degree of identity or of analogy of the sequences from papillomaviruses of different types which mutually hybridize under non-strict or non-stringent conditions must obviously be less than in the case of papillomaviruses of the same type.
The study to which the inventors proceeded has shown both that the degree of genetic heterogeneousness between diverse types of papillomaviruses was greater than previously recognized and at the same time that the different types were fond often to be associated with forms or variants of infections presenting a certain degree of specificity.
The invention consequently concerns not only the DNAs susceptible to being isolated from different new papillomaviruses which have been isolated and the probes which can be partially or entirely constituted of these DNAs, but also mixtures or xe2x80x9ccocktailsxe2x80x9d of papillomavirus types likely to be most effectively used for the diagnosis of diverse categories of infection, and of the levels of risk to the patient which accompanies the discovery of a given papillomavirus. The number of papillomavirus probes described in the present application, to which may be added, as the case may be, those constituted from the genomic DNAs of papillomaviruses which have already been previously isolated, and their associations in the specific mixtures lend greater precision to the diagnosis, notably a greater discrimination between the diverse categories of infections which may be imputed to the diverse types of papillomavirus or which may be susceptible to develop under the effect of these latter types and, within a given category of infections, to give a better prognosis of the degree of risk that these latter could be transformed to more serious disease. For example, the invention aims to provide the means permitting, in the case of infections manifesting as epidermodysplasias verruciformis, to better appreciate the degree of risk that they may evolve towards skin cancer.
In a general manner and in an attempt to simplify the following presentation, the whole genomes of the papillomaviruses will be designated by the abbreviation xe2x80x9cHPV-DNAxe2x80x9d.
With the same aim of simplification, reference will be made to appended drawings on which figure physical restriction maps of HPV-DNAs including some from previously known papillomaviruses.
These physical maps give the positions of the fractionation sites of the various restriction endonucleases. The origin of the maps is generally constituted of the site of a single cut. The distances from this point of origin are expressed as a percentage of the total length of the genome. One unit of the map represents 1% of the length of the genome.